title: A conserved proline-rich sequence between the N-terminal signal-anchor and catalytic domains is required for assembly of functional cytochrome P450 2C2.
abstract: In cytochrome P450 2C2, the region which links the N-terminal signal anchor with the catalytic domain contains a highly conserved proline-rich region with the sequence, 30-PPGPTPFP-37. Mutation of proline-30 or proline-33 diminished activities of the mutants expressed in COS-1 cells (Chen, C., and Kemper, B. (1996) J. Biol. Chem. 271, 28697-28611). Substitution of alanine, proline, or arginine for glycine-32 abolished laurate hydroxylase activity of the proteins expressed in COS-1 cells, which suggests that this residue is also functionally important. To determine the basis for the decreased activity in COS-1 cells, the activities and spectral properties of mutant proteins expressed in insect cells and bacteria were determined. Substitution of alanine for either proline-30 or -33 resulted in reduced expression in insect cells of functional cytochrome P450 hemoprotein and an increase in the expression of inactive cytochrome P420. In contrast, substitution of alanine for proline-31, -35, or -37 resulted in hemoproteins with spectra similar to cytochrome P450 2C2 so that the amount of cytochrome P450 expressed in insect cells correlated with the activities of the mutants in COS-1 cells. The laurate hydroxylase activities per nanomole of cytochrome P450 in insect microsomes were similar for wild type and all mutants, indicating that, once folded, the catalytic activity of membrane-bound cytochrome P450 was not affected by the mutations. Expression in bacteria resulted in diminished expression of cytochrome P450 for all mutants, with the greatest decrease for the proline-30 and -33 mutants, and increased cytochrome P420. In contrast to the insect cell studies, the proline-30 and -33 mutants were inactive, while the other mutants had specific activities 30-70% of cytochrome P450 2C2. These data are consistent with a role for the proline-rich region in efficient assembly of cytochrome P450 2C2 in eukaryotic cells. Mutations of this region also may affect the conformational integrity of the proteins, which was revealed by assays of solubilized bacterially expressed proteins.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Chen
authors.forename: B
authors.initials: B
authors.lastname: Doray
authors.forename: B
authors.initials: B
authors.lastname: Kemper
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D017124:Conserved Sequence / Q000235:genetics*; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011392:Proline / Q000235:genetics; D016415:Sequence Alignment; D013053:Spectrophotometry; D014162:Transfection / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
chemical_list: D003580:Cytochromes; C013464:cytochrome P420; D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D006899:Mixed Function Oxygenases; C084930:cytochrome P-450 2C2; D042926:Cytochrome P-450 CYP4A